Research and development of bubble technologies has gained immense popularity in recent times, as many industries are beginning to recognize the potential applications of bubbles at the micro- and nanoscale (Azevedo et al. 2019; Favvas et al. 2021; Paknahad et al. 2021). Despite their biphasic nature, bubble solutions, such as incompressible liquids, provide a powerful platform for gas-starved systems (e.g., aeration chambers, biological reactors, and chemical oxidative catalysis reactors). They provide these systems with a sufficient supply of gases while retaining the storage, pumping, and piping advantages of an incompressible liquid (Alheshibri et al. 2021; Yildirim et al. 2022). Furthermore, bubbles containing gases such as oxygen, carbon dioxide, and hydrogen in oil or water can be used for various purposes, such as disinfection, water quality improvement, animal and plant growth promotion, or to manufacture products such as cosmetics, fuel catalysts, drug delivery systems, and ultrasonic contrast agents (Favvas et al. 2021; Phan et al. 2020; Piotrowska et al. 2020; Singh et al. 2021a, 2021b, 2021c; Wen 2009; Zhou et al. 2021).

The generation, measurement, and characterization of microscopic bubbles were the main areas of interest in the field of bubble technology (Zhou et al. 2022). Technologies including particle aggregation, contaminants reduction, disinfection, chemical oxidation, adsorption, or biological transformation are made possible by the features of NBs, such as gas bridge, stability, and scouring, as well as the low chemical needs for NB production. NBs present chances to enhance existing technologies or enable new alternatives in order to produce fewer by-products and achieve safer water (Zodrow et al. 2017; Garcia-Segura et al. 2018). NBs' surface charge makes it possible for them to naturally adhere to emulsified, colloidal, and suspended substances, which aids in their removal from wastewater streams and improves process effectiveness (Khan et al. 2020).

More recently, nanobubbles (NBs), which are also called ultrafine bubbles, have attracted significant attention compared to microbubbles (MBs) owing to their exceptional interfacial properties, molecular scale size, and unexpected long-term stability (Li et al. 2021a, 2021b; Nirmalkar et al. 2018; Qian et al. 2018; Yildirim et al. 2022). Because MBs disappear quickly in solutions, bubbles cannot be guaranteed in the product (Lee et al. 2017; Tano et al. 2013). However, NBs can be retained in a liquid for a long time, regardless of the solubility of the gas in the solvent (Ebina et al. 2013; Meegoda et al. 2018; Nirmalkar et al. 2018; Nishiyama et al. 2016; Ulatowski et al. 2019; Wang et al. 2021; Zhang et al. 2021). This effect can be attributed to the fact that their Brownian motion is greater than their buoyancy, in addition to their highly charged surfaces on the nanometer scale (Kyzas and Mitropoulos 2021; Lim et al. 2021; Singh et al. 2020; Yasui et al. 2018; Zhou et al. 2021). Because of their special qualities, such as their high stability, large specific surface area, and transparency due to a diameter smaller than the wavelength of visible light, NBs can be used more efficiently for a variety of processes, including improving methane production in anaerobic digestion, froth flotation, waste-water treatment, visualization improvement as an ultrasound contrast agent, and food processing (Gao et al. 2017; Hu and Xia 2018; Khatun et al. 2021; Lukianova-Hleb et al. 2014; Phan et al. 2020; Singh et al. 2021a, 2021b, 2021c; Singh et al. 2021a, 2021b, 2021c; Thakur et al. 2019; Xia and Hu 2018).

In particular, with diameters less than 200 nm, NBs can be ultra-stable under various conditions, such as temperature, pH, and different salt solutions (Attard 2003, 2014; Boshenyatov et al. 2019; Koshoridze and Levin 2019; Li et al. 2021a, 2021b; Qian et al. 2018; Shukla et al. 2020; Wang et al. 2021). Their long-term stability can be attributed to several factors, including bubble size, zeta potential values, and interfacial characteristics (Hewage et al. 2021; Yang et al. 2021). However, they are not yet fully understood (Wang et al. 2020; Wen 2009; Zhang et al. 2017). In addition, the ultra-high stability of NBs can be affected by environmental factors such as the type of gas used, the amount of energy used to generate it, the type and concentration of ions, the presence of organic matter such as surfactants and ion type and concentration, and physical conditions such as temperature, pressure, and pH (Agarwal et al. 2011; Rodriguez-Rodriguez et al. 2015; Ulatowski and Sobieszuk 2020). More importantly, at zeta potential values for diameters below 200 nm, dense repulsive forces are formed between individual bubbles. Consequently, NBs can exist for months without fusion, even in liquids (Hirsch et al. 2013; Zhang et al. 2020). However, most studies on the stability of NBs focus only the size and surface charge (Alheshibri et al. 2016; Xiong et al. 2021).

Since the absolute amount of NBs is inherently less than that of MBs for use in storage and transit, it is important to take their number or concentration into account when applying them in various applications (Abenojar et al. 2020; Wu et al. 2019; Xiong et al. 2021). Recently, Kanematsu et al. (2020) published the long-term stability of NBs according to the interaction between the polymer and the NBs by observing the concentration change, but for utilization of NBs, the concentration change of NBs via various conditions should be studied.

The nanoparticle tracking analysis (NTA) technique is specifically aimed at detecting and analyzing these relatively low concentration structures of extremely small size (compared with that of 'standard' bubbles) (Nirmalkar et al. 2018). NBs can be characterized by directly visualizing nanoscale particles in suspension (40–1000 nm) with high resolution in real time, owing to their unique NTA capability (Grabarek et al. 2019; Porter et al. 2020). Using single-particle tracking techniques, NTA can identify a particle's size from its diffusion in a Brownian motion system. The size distribution of the particles can also be identified thanks to the tracking of multiple particles. The laser light is scattered by the particles in its path and is easily collected by the microscope objective for viewing using a digital camera. The Stokes Einstein equation is used by NTA software to calculate the hydrodynamic diameters of numerous particles after it simultaneously analyzes each particle separately (Grabarek et al. 2019; Vogel et al. 2021).

Effective implications require an understanding of the characteristics and behavior of these bubbles. Nonetheless, it is considered that NB behavior is complex. Hence, a thorough investigation is needed to properly understand how the generation process, concentration, and external influences on NB stability are impacted. In this manner, the ultra-high stability of NBs was studied by observing the concentration and size changes of high-concentration NBs under various conditions, such as aging, temperature, centrifugation, shaking, and stirring. The presence of NBs in a solution for 120 days after generation demonstrates that NBs has significant potential for use in commercialized products. This study provides a new method for NBs to be put to practical use in various industries.

Results and discussion

NBs have attracted much attention due to their countless uses in many scientific and technological fields. Various commercially available generators have been used to produce NB water; however, they are difficult to generate at high concentrations. A novel, previously patented device was recently developed that can produce a high concentration of NBs in the aqueous solution required for this study (Park et al. 2022; Park 2022). Using this device, the NBs were created by continuously passing water and air through a generator, and various conditions were applied to the NBs prior to analysis using NTA (Scheme 1). The generator used in the study produces NBs when the gas–liquid mixture collides with the structure in the generator owing to the power received from the pump. The diaphragm pump is capable of circulating a fluid at a rate of 7.5 L/min at 25 psi. The fluid moves through the pump along a tube and passes through an NB generator with a structure that rotates the fluid. The fluid normally moves in a straight line along the pipeline with the pump; however, in this structure, it moves spirally, thereby increasing the contact area with the wall. Finally, the irregular surface on the inner wall of the NB generator obstructs the flow of fluid, resulting in frequent collisions and formation of NBs. The number of NBs can be controlled by adjusting the operating time of the device, and the manufacturing time can be reduced by simultaneously connecting several NB generators. Generated NBs were stored in airtight glass vial to prevent contamination.

Scheme 1
scheme 1

Schematic illustration of NB generation and analysis of their stability after exposure to various conditions

After NB generation, all solutions were analyzed using NTA, which scatters light by irradiating the particle-scattered solution with laser, and the scattered light is visualized using a digital camera. Scattering faculae were produced when the laser light collided with the particle. The high-speed camera captured the trail of scattered faculae. For accurate analysis and comparison, the appropriate concentration (10–30 particles visible on the screen of the program) was selected by dilution, and the light intensity was adjusted using Slider Shutter (1500) and Slider Gain (330) (Fig. 1a). The concentrations of NBs after 1, 2, 3, 7, 8, 9, and 12 h of production were 0.53, 0.92, 1.45, 2.03, 2.43, 2.5, and 2.37 billion/mL, respectively, as shown in Fig. 1b. The size range of the NBs was approximately 100 nm; it showed limited change with time, indicating that the production time does not affect the physical properties of the NBs, but only their concentration (Fig. 1c). To confirm that these numbers only originated from NBs and not impurities, such as dust, the bubble solution was lyophilized and redispersed in water. Similar to pure water, a small percentage of bubbles was observed, indicating a high NB concentration (Fig. 1d).

Fig. 1
figure 1

a A NB image analyzed through NTA, b concentration and c size of NBs according to production time, and d concentration change of NBs after freeze drying and redissolving

Long-term stability through changes in the size and surface charge of NBs has been measured using various methods in different types of studies, and NBs have been reported to be stable for several months. However, the temporal change in NB concentration required for commercialization, along with mass production and distribution, remains unknown. In our study, the change in concentration of NBs was observed for 120 days using the prepared NBs; the final concentration of NBs was approximately 81% of the initial concentration. The NBs were stable for a long time, with negligible changes within the margin of error that occurred during observation. The average size of the initially prepared NBs was 96 nm, and the largest size was 117 nm after 20 days. However, after 120 days, the average size was 101 nm, which was not significantly different from the initially measured size (Fig. 2a). As shown in Fig. 2b, a significant change in the distribution mode was not observed over time, but NBs which have over 150 nm size were temporarily observed after 20 days. The temporary increase in the NB size and distribution mode can be attributed to the merging of unstable NBs and Ostwald ripening (Antonio Cerron-Calle et al. 2022; Yaparatne et al. 2022). After the merged NBs disappeared from the solution, the average size and distribution mode decreased again.

Fig. 2
figure 2

Changes in a concentration, size and b size distributions of NBs over time

Generally, temperature change affects the volume of the gas. Thus, the stability of NBs at various temperatures was observed to determine the effect of these volume changes on the concentration and size of the NBs. NBs were stored at 5 °C, 25 °C, 60 °C, and 80 °C for 4 months, and the concentrations of NB at the end of the experiment were confirmed to be approximately 85.7, 81.0, 103.0, and 84.8% of the initial NB concentration, respectively (Fig. 3a). In addition, the size of the NBs at all temperatures did not differ significantly from that of the initially generated NBs (Fig. 3b). Generally, bubbles disappear in three ways: buoyancy, dissolution, and agglomeration. First, the rise in temperature can make the NBs move faster, which can lead to forced buoyancy. However, it also increases Brownian motion, which improves their retention in the solution. Second, because the solution was already saturated with gases during NB production, the internal gases in the NBs struggled to dissolve in the solution. Finally, the high charge density around NBs with diameters less than 200 nm is sufficiently high and stabilizes the NBs. Furthermore, the repulsive forces between the bubbles prevent coalescence. Owing to these phenomena, a solution of sub-200-nm NBs can be stably stored at various temperatures.

Fig. 3
figure 3

a Concentration and b size change of NBs depending on the temperature

NB solutions were also exposed to various conditions that may occur during transportation or be introduced into production, including centrifugation, shaking, and agitation. Initially, the prepared NB solution was stimulated at 18,800 RCF using a centrifuge for intervals of 30, 60, and 90 min. The concentration of NBs was maintained above 90% of the initial concentration, regardless of the operating time of the centrifugation, and no significant change in size was observed (Fig. 4a). The NBs larger than distribution mode disappear over time due to the merging of unstable NBs and Ostwald ripening (Fig. 4b).

Fig. 4
figure 4

a Concentration, size and b size distributions change of NBs after centrifugation at 18,800 RCF for a total of 90 min

The NBs were stimulated using a shaker for 8 h to create severe physical impacts, such as collisions between NBs and bottle surfaces or NBs, in order to further demonstrate their ultrahigh stability. As shown in Fig. 5a, the concentration of NBs was maintained at 96% or more of the initial concentration, and the NB size was constant at approximately 100 nm. In the shaking experiments, as in the centrifugation experiments, decrease of NBs was observed over time. Meanwhile, a decrease in the distribution mode was observed temporarily, but no clear trend was observed in the distribution mode (Fig. 5b). In addition, the stability was further confirmed by stirring for 8 h; the concentration of NBs was almost similar to the initial concentration, and the size remained almost constant between 90 and 100 nm (Fig. 6a). NBs with a size larger than the distribution mode disappeared, and a trendless change was observed in the distribution mode (Fig. 6b). A concentration higher than the initial concentration after 8 h of stirring and a temporary change of distribution mode is attributed to the NTA measurement error.

Fig. 5
figure 5

a Concentration, size and b size distributions change of NBs after shaking at 1500 rpm for a total of 8 h

Fig. 6
figure 6

a Concentration, size and b size distributions change of NBs after stirring

In our study, a high concentration of NB water (approximately 2 billion cell/ml), which could be produced without the use of a surfactant, was analyzed to assess the ultra-high stability of NBs less than 200 nm in diameter. When a surfactant is present, the stability may vary depending on its concentration, type, and charge, and this phenomenon can be studied under more specific conditions and methods in the future. Despite the fact that a number of experiments were conducted, which provided light on the stability of NBs, diverse NB experiments frequently point to diverse stabilization mechanisms. Therefore, further studies are needed to have a better understanding and establish a solid theory.


Understanding the unique and promising properties and behavior of NBs is essential for a variety of industrial applications. Therefore, a series of laboratory experiments were conducted. In particular, for NBs with diameters less than 200 nm, ultra-high stability is required for to be able to use NBs in mass production and distribution. We successfully produced NB water at concentrations above 2 billion/mL and approximately 100 nm in size to test the stability of the bubbles. To estimate the ultra-high stability, the concentration and size change of the manufactured NB water were observed after various temperature changes or physical shocks were applied using a centrifuge and a shaker. After storage at various temperatures, the NBs were stable without significant changes in number and size for 120 days. Furthermore, they showed high stability when exposed to various physical impacts, such as centrifugation, shaking, and stirring. These experimental findings show that the high stability of sub-200-nm NBs has tremendous application potential in NB research and development, creating a new avenue for bubble technologies.

Materials and methods


Tertiary distilled water (DW) (Expe-CB VF90, MIRAEST CO., LTD) was used as the solvent. Fresh DW was collected in a 100-L tank and allowed to reach equilibrium with the atmospheric gases at room temperature for 24 h. NTA (NanoSight NS300, Malvern), shaker (Vortemp 56, Labnet International, Inc.), centrifuge (LABOCENE 1736R, HANIL SME Co., Ltd), and hotplate stirrer digital control (MSH-20D, Daihan Scientific Co. Ltd.) were used to maintain the conditions for stability analysis of the NBs.

Generation of high-concentration NBs

NBs were produced by placing 1 L of DW in a tank and continuously passing it through a custom-made NB generator A using a diaphragm pump. Air was injected for the first 5 min, and the device was operated to remove the other impurities. NB water was produced by confining air in a storage tank and continuously using it. The concentration of bubble water was adjusted according to the operating time of the device, and the production time was controlled by simultaneously installing additional devices at the same time.

Characterization of NBs

NTA device was used to measure the number of NBs in the produced NB solution, and a laser with a wavelength of 532 nm was used. For consistent measurement, the same values of Slider Shutter, Slider Gain, and Detect Threshold, which are related to camera level, were used as 1500, 330, and 5, respectively.

Analysis of the stability of NBs depending on the temperature

To confirm the reaction to heat, NB water was stored at 5, 25, 60, and 80 °C. For storage, a 10-ml injection vial with a 20-mm aluminum cap was used, and 4 ml of NB water was added and sealed. In addition, a refrigerator and oven with each temperature set were used as devices to maintain the appropriate temperature. The number of NBs over time was measured using NTA.

Stability test of NBs using centrifugation

Using a centrifuge, the concentration change of NB water was measured under 18,800 RCF for 90 min. The sample used in the centrifuge was 10 ml of NB water in a 15-ml Falcon tube.

Stability analysis of NB using shaker

Using a shaker, the concentration change of the NB water with time was measured under two different rpm conditions. A shaker speed of 1500 rpm was used, and the concentration change was observed by operating it for a total of 8 h. Samples were tested by placing 1 ml of NB solution in a 2-ml Eppendorf tube.

Analysis of the stability of NB under stirring

The experiment was performed using a hotplate stirrer digital control. The NB solution (20 mL) was placed in a 30-mL vial and stirred at 300 rpm for up to 8 h.